Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

Area of Science:

• Structural Biology
• Biophysics
• X-ray Crystallography

Background:

• Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) is crucial for determining structures of challenging proteins.
• Current SFX methods waste up to 99% of sample due to the pulsed nature of XFELs, limiting structural studies.
• Efficient sample delivery is essential for maximizing data collection and minimizing sample consumption in SFX.

Purpose of the Study:

• To improve sample conservation in SFX experiments by enhancing injection methods.
• To develop a sample-saving technique compatible with existing XFEL infrastructure.
• To demonstrate an electrically triggered segmented flow approach for SFX sample delivery.

Main Methods:

• Utilized 3D-printed injection devices with gas dynamic virtual nozzles (GDVNs).
• Integrated metal electrodes to induce electrowetting for precise droplet generation control.
• Employed an electrical feedback mechanism to synchronize droplet release with XFEL repetition rates.
• Tested the improved method with lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase microcrystals.

Main Results:

• Achieved improved control over droplet generation frequency using electrowetting.
• Demonstrated enhanced sample conservation compared to continuous GDVN injection.
• Successfully applied the electrically triggered segmented flow approach at the LCLS Macromolecular Femtosecond Crystallography instrument.
• Validated the method's effectiveness for challenging protein microcrystal studies.

Conclusions:

• The electrically triggered segmented flow approach significantly improves sample utilization in SFX.
• This method offers a revolutionary solution to the sample consumption problem in XFEL-based structural biology.
• The enhanced injection technique is compatible with current XFELs and advances structural determination capabilities.